Metabolic engineering incorporates a variety of academic disciplines to generate high production systems for desired, and largely commercial, products. Many of these products are expensive to produce because the downstream processing and purification is often very complex. High value products such as succinate, ethanol, and lactate are produced from glucose in Escherichia coli via the anaerobic central metabolic pathway. Additionally, heterologous genes are often expressed in E. coli to produce valuable compounds such as polyketides, nutritional compounds, and pigments.
To practice metabolic engineering, the physiology of the species must be understood to determine which manipulations to pursue. Manipulations necessary for a desired product(s) must be chosen systematically and accurately. Deleting certain pathways may be fatal to the cell, while deleting others may lead to a metabolic bottleneck and shortage of immediate metabolites necessary for the desired product. Understanding these manipulations to maximize production and reduce metabolic burden on the host cell is very important.
Recent work has been done to metabolically engineer E. coli to produce isoamyl acetate, a compound in the ester family (2, 10, 19, 20, 21). Isoamyl acetate is a valuable chemical used as an industrial solvent, plasticizer, cleaner, and a solvent for lacquer coatings and nail polish. However, its most important use is in the food industry where 74,000 kg/year are used (7), largely because it is a key element in the flavor of sake.
Two alcohol acetyltransferases (ATF1 and ATF2) in Saccharomyces cerevisiae were found to produce isoamyl acetate from acetyl-CoA and isoamyl alcohol during the yeast fermentation process in sake wine as well as other wines and beers (3, 15, 23, 24). Several constructs expressing ATF2 on a high-copy number plasmid were prepared and used in our laboratory for microbial, normative production of the ester in E. coli (19, 20).
While most focus has been on producing isoamyl acetate aerobically, pathway manipulations have also been applied to increase isoamyl acetate production anaerobically (21). As seen in FIG. 1, the ethanol (adhE) and acetate (ackA-pta) production pathways compete directly with the ester pathway for the precursor acetyl-CoA. Additionally, the lactate (ldhA) and succinate pathways compete indirectly with the ester pathway for the precursor pyruvate. Anaerobic cultures of a strain with a mutation in ackA-pta, thereby eliminating acetate production, produced more ester than the parent strain, demonstrating the positive effect of channeling carbon flux away from acetate production. However, eliminating lactate production with an additional mutation in ldhA resulted in a surprising drop in ester production. The cells must always maintain a proper redox balance between the cofactor pair NADH and NAD+, and therefore the ethanol production pathway was restored, diverting carbon flux from ester production.
Another strategy for producing large amounts of isoamyl acetate anaerobically is to overexpress the succinate-producing pathway and eliminate the ethanol-producing pathway. Because the volatility of isoamyl acetate and succinate differs greatly, the two could be easily separated in an industrial setting. By employing this strategy, the cells remain healthy with a proper redox balance, and in the process two valuable and easily separated compounds are produced.
Succinate is valuable as a precursor to numerous products in the pharmaceutical and chemical industries (9). Two major pathways produce succinate (FIG. 1). The fermentative pathway anaerobically converts OAA to malate, fumarate, and finally succinate, reducing 2 moles of NADH (17). Aerobically, the glyoxylate pathway converts 2 moles of acetyl-CoA and 1 mole of OAA to 1 mole each of succinate and malate (12), which can be further converted to succinate via the pathway described above.